Peripheral Blood RNA Research Articles

Human Immunodeficiency Virus Type 1 (HIV‐1)‐Specific T Cell Responses Correlate with Control of Acute HIV‐1 Infection in Macaques

Macaca nemestrina efficiently control acute human immunodeficiency virus type 1 (HIV-1) infection. The roles of helper (Th) and cytotoxic (CTL) T cells in controlling acute HIV-1 infection in both peripheral blood and lymph node mononuclear cells (PBMC and LNMC) were assessed in this model. Th and CTL responses to HIV-1 were detected within 2 weeks following HIV-1 infection, and CTL responses to HIV-1 antigens peaked at 4 weeks after infection (>100 HIV-specific CTL/10[6] PBMC), coincident with reductions of HIV-1 RNA and DNA levels in peripheral blood. HIV-1-specific Th and CTL were present in LNMC 6 weeks after infection. Although HIV-1 antibodies were detected 2 weeks after infection, maximal HIV-1 antibody responses were not generated until > 13 weeks after inoculation. Thus, T cell responses temporally correlate with control of HIV-1 in macaques. The induction of a brisk HIV-1-specific CTL response may have been facilitated by a persistent Th response.

Distribution of hepatitis C virus (HCV) RNA in whole blood and blood cell fractions: plasma HCV RNA analysis underestimates circulating virus load.

Previous experiments using a cationic surfactant to detect hepatitis C virus (HCV) RNA in whole blood (WB) suggested that WB was a more plentiful source of viral RNA than was plasma. The relative HCV RNA titers in WB, plasma, peripheral blood mononuclear cells (PBMC), neutrophils, and red blood cells (RBC)/platelets from 10 patients with chronic HCV infection were compared. WB contained significantly more HCV RNA than plasma, which contained more HCV RNA than PBMC, neutrophils, or RBC/platelets (P < .001). To determine if this increased sensitivity was clinically relevant, results of WB and plasma HCV RNA assays were compared with commercial quantitative and qualitative plasma HCV RNA assay results obtained for patients receiving interferon therapy. WB was significantly more sensitive than commercial plasma reverse transcription-polymerase chain reaction for detecting HCV RNA (P < .005). These data indicate that a significant proportion of HCV RNA in peripheral blood is not identified by standard plasma RNA detection methods.

Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests.

Reverse transcription-polymerase chain reaction was used to identify hepatitis C virus (HCV) RNA in peripheral whole blood (WB) and plasma samples from 15 patients with chronic, unexplained hepatitis. These patients were serologically negative for hepatitis A, B, and C and were classified as having chronic non-A, non-B, non-C hepatitis (NANBNC). HCV RNA was repeatedly detected in WB samples from 10 (67%). In contrast, plasma samples from only 5 were intermittently positive. Statistically, HCV RNA detection in WB was significantly more sensitive than in plasma. Nucleic acid hybridization and HCV genotypic analysis confirmed the specificity of the HCV RNA assay. Liver biopsies from these patients suggested histopathologic differences between HCV RNA-positive and -negative groups. These data demonstrate that HCV infection is present in patients with unexplained chronic hepatitis more frequently than previously believed. Additionally, WB HCV RNA detection is more sensitive than plasma assays in identifying antibody-negative HCV infection.

Familial Erythrocytosis Associated With a Short Deletion in the Erythropoietin Receptor Gene

AbstractFamilial erythrocytosis (familial polycythemia) inherited as an autosomal dominant trait has recently been reported to be associated with mutations in the gene encoding the erythropoietin receptor (EpoR) in a small number of families. We studied a new kindred with dominantly inherited familial erythrocytosis associated with heterozygosity for a deletion of seven nucleotides between positions 5985 and 5991 in exon 8 of the EpoR gene, resulting in an EpoR peptide that is truncated by 59 amino acids at its C-terminus. A 7-bp direct repeat is present in the normal EpoR gene at the site of this mutation, consistent with the slipped mispairing model for the generation of short deletions during DNA replication. Hypersensitivity to Epo of erythroid progenitors from an affected individual was observed in in vitro methylcellulose cultures, as indicated by more numerous and larger colonies compared with those of a control subject. To study mutant EpoR function, the cDNA encoding the mutant EpoR was synthesized by reverse transcription-polymerase chain reaction of peripheral blood RNA from the proband and stably tranfected into murine interleukin-3–dependent 32D cells. Epo dose-response assays showed that cells expressing the mutant EpoR displayed fivefold to 10-fold increased sensitivity to Epo compared with cells expressing similar numbers of the wild-type EpoR.

Peripheral Blood Tyrosinase Messenger RNA Detection and Survival in Malignant Melanoma

The most widely accepted criteria for the evaluation of prognosis of malignant melanoma are histopathologic and clinical presentation. No currently available laboratory tests provide additional prognostic information. It has recently been suggested that reverse transcription and polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood might be useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. To further evaluate the clinical relevance of this potential marker, we examined peripheral blood samples from patients with malignant melanoma in different stages of disease for the presence of tyrosinase mRNA. Total cellular RNA was extracted from heparinized peripheral blood cells from 64 patients with malignant melanoma, from five healthy control subjects, and from four patients with other cancers using the RNAzol A method. For analysis of tyrosinase mRNA, RT-PCR was performed as previously described by Smith et al.; the sensitivity of this assay was tested using RNA extracted from human melanoma cells (SK-mel 1 and SK-mel 3 cell lines) serially diluted with peripheral blood obtained from healthy control subjects. Two additional human melanoma cell lines (SK-mel 30 and RPMI-7951) served as positive controls for RT-PCR detection of tyrosinase mRNA. Overall patient survival curves were constructed using Kaplan-Meier estimates. Tyrosinase mRNA was detected by RT-PCR assay of all four of the established melanoma cell lines tested. Nine of the 64 patients with malignant melanoma were found to have detectable tyrosinase mRNA in their peripheral blood cells (tyrosinase-positive patients). The 16 patients with localized primary melanoma did not have detectable tyrosinase mRNA in their peripheral blood cells. Among the 48 patients with metastatic disease, all 27 patients who exhibited no evidence of disease progression were tyrosinase negative. Notably, all nine tyrosinase-positive patients had visceral metastases and were found to exhibit disease progression at the time of the sampling. Four of the nine tyrosinase-positive patients were also found to test negative at times without evidence of progressive disease; one patient became negative after achieving stable disease and three became positive for tyrosinase transcripts on disease progression. The probability of survival from time of sampling was significantly lower in the nine tyrosinase-positive patients when tested versus the 23 patients with comparable disease but without detectable tyrosinase mRNA (two-sided; P < or = .05). The results of this study demonstrate that the detection of tyrosinase mRNA in cells in the peripheral blood by RT-PCR may be a useful prognostic marker for predicting tumor progression and poor clinical outcome in patients with malignant melanoma.

P-69 Study of perinatal HCV transmission - expression and persistence of HCV RNA in infants' peripheral blood

P-69 Study of perinatal HCV transmission - expression and persistence of HCV RNA in infants' peripheral blood

α-Fetoprotein messenger RNA in peripheral blood as a marker of circulating hepatocellular carcinoma cells

AFP mRNA was detected in the peripheral blood of patients with hepatocellular carcinoma (HCC) by two-step polymerase chain reaction (PCR) preceded by reverse transcription to elucidate whether the existence of AFP mRNA in the blood could be helpful for clinical detection of metastasis of HCC. AFP mRNA in the blood was positive in eight of 21 patients with HCC (38.1%). The three HCC patients with distant metastasis were among the eight patients with detectable mRNA. Serum AFP level was more than 400 ng/ml in five of the eight HCC patients with mRNA in the blood, but less than 100 ng/ml in 11 of the 13 without detectable mRNA. In contrast, AFP mRNA in the blood was detected in only one of 32 patients without HCC (3.1%); these latter consisted of 10 patients with chronic hepatitis, six with liver cirrhosis and 16 with non-liver disease. The AFP mRNA-positive patient without HCC had chronic hepatitis type C, whose serum AFP concentration was 4 ng/ml. These data suggest that AFP mRNA may derive from AFP-producing HCC cells in the blood and that its existence may be helpful for clinical detection of metatasis of HCC. However, further study is necessary to determine what the AFP mRNA detected in the blood of the chronic hepatitis patient is derived from.

Molecular characterization of glycophorin A transcripts in human erythroid cells using RT-PCR, allele-specific restriction, and sequencing.

Glycophorin A (GPA) is an erythroid-lineage-specific membrane sialoglycoprotein which occurs in two allelic forms, M and N, which form the antigens of the MN blood group. Purified cDNAs and RNAs isolated from peripheral blood and erythroleukemia cell lines, HEL and K562, were used to develop an RT-PCR technique for amplifying GPA gene transcripts (GYPA). The relative expression of transcripts from the M and N alleles was determined using restriction analysis of these amplified products with four allele-specific restriction endonucleases. The use of this method permits the sensitive identification of GYPA transcripts in these cells and confirms GPA protein expression in the erythroleukemia cell lines and the MN phenotypes of individuals determined by immunolabeling with GPA allele-specific monoclonal antibodies. A novel restriction pattern was obtained using peripheral blood RNA from two individuals with a rare inherited variant allele, GPA Mg. Sequencing of the cDNA obtained using this method revealed a single C to A transversion in the fourth codon in the mature GYPA N coding sequence is responsible for the difference between GYPA Mg and GYPA N.

Limites du diagnostic immunosérologique et moléculaire de l'hépatite C

Hepatitis C is the most common cause of post-transfusion hepatitis, as well as of the viral chronic liver disease in the western world. However since it is even more often asymptomatic than HBV, this is not truly recognized. The detection of hepatitis C can only rely on serological and virological methods and require their extensive use in screening programs. Following the molecular identification characterisation of HCV, it became possible to detect virus specific antibodies. The first generation Elisas were limited in their scope and have been replaced by second and third generation tests with better sensitivity and specificity. These assays detect antibodies to several sets of HCV protein including the C22 core, the C33 and C100, which correspond to the non structural regions (NS3 and NS4 respectively). More recently, NS5 proteins have also been added and synthetic peptides have replaced some of the recombinant proteins used initially. In spite of improved sensitivity and specificity, last generation Elisas still require confirmation by supplemental assays which can be of different types (immunoblot or combined Elisas) and include sets of structural and non structural recombinant proteins or peptides. New tests are needed to improve sensitivity and proficiency of this mandatory confirmation procedure. It is unclear at this stage whether the dogma inherited from HIV to request two sets of reactive antibodies will be also warranted by experience in HCV infection. The biggest limitation of present HCV tests is the delayed appearance of anti-HCV following primary infection. Even more worrisome is the fact that 10% of chronic infection with liver disease still remain seronegative, despite circulating HCV RNA in serum and/or liver as well as expressing HCV antigen demonstrable in liver tissue by immunostaining. Such a proportion is even more common in settings with immune deficiencies including organ transplantation and HIV infection. DNA amplification methods, such as PCR or others, must be used in order to demonstrate HCV RNA in combination with reverse transcription steps. This new powerful technology must be however applied under stringent quality control procedures and cannot be yet considered for screening or routine diagnosis although it can detect viremia as early as a week after exposure and help to monitor interferon treatment. During acute hepatitis, the delay in the appearance of anti-HCV hampers acute phase diagnosis. The early detection of HCV RNA in peripheral blood, confirms the diagnosis and opens up therapeutic possibilities. In chronic hepatitis, the diagnosis of seronegative forms may only be resolved by PCR. Moreover, the presence of HCV RNA in peripheral blood represents the only marker of on going viral replication and coincides with the severity of liver damage. During treatment with interferon, the follow up of HCV RNA sequences makes it possible to monitor its efficacy. The search for HCV RNA sequences directly in liver tissue shows that HCV may replicate in the liver in the absence of viremia. The presence of HCV RNA in the liver and the serum of liver transplanted patients is essential for the etiological diagnosis and management of hepatitis and bone marrow failure occurring after transplantation. Epidemiological study using PCR is a major tool in documenting vertical transmission between mother and child. Finally, PCR is important for the analysis of the HCV genome. Thus, in France there are at least three main strains, one close to the US prototype, the other close to the Japanese strain, possibly responsible for a more severe illness, and a third one distinct from the previous two. Two major HCV genotypes, F1 and F2, corresponding to HCV type I and II (USA prototype and Japanese) with prevalence of 45% and 55% respectively, were found in France. F1 infected patients were younger and more often male than F2 group. Nine of 28 patients in F1 genotype infected group had history of drug abuse but none i

Variability in the amount of β-globin mRNA in β0 thalassemia

Globin mRNA isolated from a number of β 0 thalassemia patients of different ethnic origins was analyzed by RNA-cDNA hybridization and, in two cases, by fingerprint analysis of 125I-labeled mRNA. Quantitation of the relative amounts of α- and β-mRNA by hybridization to purified α- and β-cDNA revealed that in approximately half the cases, there was <1% as much β-mRNA as α-mRNA. In the rest of the cases, low levels of β-like mRNA were detected in amounts 4–12% as abundant as α-mRNA. There was variability in the yield of β-like mRNA in patients of the same racial group, in the same patient at different times and in similarly affected siblings: β-mRNA was virtually absent in some samples, whereas low but significant levels were found in other samples. In one patient, β-like mRNA was not detected in peripheral blood RNA, but was present in the RNA of bone marrow cells. In one case, the thermal stability of the β 0 thalassemia mRNA-β-cDNA hybrid was measured and found to be slightly lower than that of the authentic β-mRNA-β-cDNA hybrid. In none of the cases tested was there synthesis of β-globin chains directed by β 0 thalassemia mRNA in a cell-free protein-synthesizing system, even when β-like mRNA was detected in the sample by hybridization assays. mRNA from two patients was labeled in vitro with 125I, digested with T1 RNAase and fractionated in two dimensions. Analysis of the resulting fingerprints revealed the presence of prominent α chain-specific oligonucleotides without detectable β chain-specific oligonucleotides, and thereby confirmed the results of hybridization assays showing absent or very low levels of β-mRNA in the same RNA samples. Our results support the concept that β 0 thalassemia is heterogeneous in its molecular basis even within the same racial group: in some patients, it is associated with absent β globin mRNA, whereas in other patients, it is associated with low but significant levels of nonfunctional β or β-like globin mRNA. The variable amounts of β-like mRNA detected in different samples from the same patient, and in patients with the same genotype, indicate that as yet undefined factors can influence the yield of β-like mRNA observed in β 0 thalassemia.